The long term objective of our research project is to gain insight into the regulatory mechanism for DNA replication initiation in eukaryotes using yeast as our model system. The regulation of DNA replication initiation is an important aspect of the regulation of cell growth. The goal of this proposal is fundamental in nature but may have useful applications to the control of the growth of tumorigenic cells. In yeast, specific DNA sequences known as autonomously replicating sequences (ARSs) have been isolated. These ARSs are believed to be the initiation sites for DNA replication on chromosomes. We examined the mechanism of replication initiation at these sites using three different approaches. First, we identified genes that affect the function of ARSs on minichromosomes by the isolation of minichromosome maintenance defective (Mcm) mutants. We then cloned and sequenced these genes. We also examined the biochemical function and properties of the products of these MCM genes. Second, we isolated proteins that interact directly and specifically with ARSs. Third, we analyzed the functional sequence of ARSs with reference to these mutants and the ARS-binding proteins (ABPs). Our specific aims for the next funding period will be as follows: (1) We will continue to study the biochemical and biological functions of three MCM gene products, MCM1 gene products, MCM1, MCM2 and MCM3. Special attention will be given to the analysis of the MCM1 gene product which has been shown to be a transcriptional regulator. The relationship between its role in minichromosome maintenance and transcriptional activation of mating-type specific genes will be investigated. (2) We will further define the sites of action of the MCM gene products at ARSs by direct DNA binding studies. Deletion analysis of ARSs in the mcm mutants has already implicated that the site of action of the MCM gene products is on the 5' side of the ARS consensus sequence. (3) We will look for additional ABPs that would bind to the 5' side of the ARS consensus sequence.